Qin et al. Neural Development (2017) 12:13 DOI 10.1186/s13064-017-0090-5

RESEARCH ARTICLE Open Access Septal contributions to olfactory bulb interneuron diversity in the embryonic mouse telencephalon: role of the gene Gsx2 Shenyue Qin1,2, Stephanie M. Ware5, Ronald R. Waclaw1,4 and Kenneth Campbell1,3*

Abstract Background: Olfactory bulb (OB) interneurons are known to represent diverse neuronal subtypes, which are thought to originate from a number of telencephalic regions including the embryonic dorsal lateral ganglionic eminence (dLGE) and septum. These cells migrate rostrally toward the OB, where they then radially migrate to populate different OB layers including the granule cell layer (GCL) and the outer glomerular layer (GL). Although previous studies have attempted to investigate regional contributions to OB interneuron diversity, few genetic tools have been used to address this question at embryonic time points when the earliest populations are specified. Methods: In this study, we utilized Zic3-lacZ and Gsx2e-CIE transgenic mice as genetic fate-mapping tools to study OB interneuron contributions derived from septum and LGE, respectively. Moreover, to address the regional (i.e. septal) requirements of the homeobox gene Gsx2 for OB interneuron diversity, we conditionally inactivated Gsx2 in the septum, leaving it largely intact in the dLGE, by recombining the Gsx2 floxed allele using Olig2Cre/+ mice. Results: Our fate mapping studies demonstrated that the dLGE and septum gave rise to OB interneuron subtypes differently. Notably, the embryonic septum was found to give rise largely to the calretinin+ (CR+) GL subtype, while the dLGE was more diverse, generating all major GL subpopulations as well as many GCL interneurons. Moreover, Gsx2 conditional mutants (cKOs), with septum but not dLGE recombination, showed impaired generation of CR+ interneurons within the OB GL. These Gsx2 cKOs exhibited reduced proliferation within the septal subventricular zone (SVZ), which correlated well with the reduced number of CR+ interneurons observed. Conclusions: Our findings indicate that the septum and LGE contribute differently to OB interneuron diversity. While the dLGE provides a wide range of OB interneuron subtypes, the septum is more restricted in its contribution to the CR+ subtype. Gsx2 is required in septal progenitors for the correct expansion of SVZ progenitors specified toward the CR+ subtype. Finally, the septum has been suggested to be the exclusive source of CR+ interneurons in postnatal studies. Our results here demonstrate that dLGE progenitors in the embryo also contribute to this OB neuronal subtype. Keywords: Neurogenesis, Neuronal specification, Olfactory bulb, Septum,

* Correspondence: [email protected] 1Divisions of Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA 3Neurosurgery, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Qin et al. Neural Development (2017) 12:13 Page 2 of 14

Background LGE and septum, is essential for the normal generation Olfactory bulb (OB) interneurons represent a highly of CR+ and parvalbumin+ OB interneurons [20, 21]. An- diverse neuronal population that serve as important other transcription factor Tshz1 is required components in the relay of olfactory signals from the en- for the normal generation of CB+ interneurons as well as vironment to the brain [1]. They are largely inhibitory the radial migration of multiple subtypes after neuro- and modulate local projection neuron activity by blasts ultimately reach the OB [22]. Finally, the TH+ releasing gamma-aminobutyric acid (GABA) [2, 3]. The subpopulation requires both Pax6 and Er81 (Etv1) for complex functions of OB interneurons are accomplished their normal generation [2, 23, 24, 25, 26]. While these by their high diversity, which, at least in part, can be transcriptional regulators are expressed in the migrating recognized as subtypes based on distinct biochemical and differentiating neuroblasts, the homeobox gene markers being expressed [3, 4]. In addition, OB inter- Gsx2 is highly expressed by progenitor cells in the LGE neurons occupy distinct layers of the OB, allowing them and septum, and has been shown to be critical for the to exert their functions through building connections normal generation of many OB interneuron subtypes selectively with tufted cells or mitral cells, the major [20, 27, 28, 29, 30, 31, 32]. Specifically, it was demon- projection neurons in the OB [5, 6, 7]. Interestingly, OB strated that the generation of OB interneurons is se- interneurons of different subtypes show varied prefer- verely compromised when Gsx2 is absent in the dorsal ences in layer localization and neuronal connectivity [3]. LGE (dLGE) [31]. Gsx2 is also highly enriched in the VZ For example, tyrosine hydroxylase-labeled (TH+) progenitor cells of the septum, which represents another dopaminergic interneurons and calbindin+ (CB+)in- important source of OB interneurons at perinatal time terneurons are enriched in the glomerular layer (GL), points [2, 7, 9, 10, 14, 33]. Gsx2 is required for normal whereas calretinin+ (CR+) interneurons are found in gene expression in the embryonic septum, including its both GL and granule cell layer (GCL). The specific downstream effector Ascl1 and related targets [34]. roles of the diverse OB interneuron subtypes in olfac- However, the function of Gsx2 in the specification of tory circuits is not well defined, however, studies have septum-derived OB interneurons has not been shown these interneurons originate from the embry- examined. onic ventral telencephalon and regional progenitor In this study, we utilized two genetic fate-mapping domains in the postnatal SVZ [2, 8, 9, 10]. tools to investigate the LGE and septal contributions to Unlike the locally born projection neurons [11, 12, 13], OB interneuron diversity. In addition, by using a condi- OB interneurons are generated caudal to the bulb within tional knockout strategy, we examined the role of Gsx2 the ventral telencephalon, largely the lateral ganglionic in the generation of septum-derived OB interneurons. eminence (LGE) and septum, from embryonic day 12 Our data demonstrate that the LGE and septum give rise (E12) until birth and subsequently from the postnatal to OB interneuron subtypes differently, with the LGE and adult SVZ, which represents the derivative of these being heterogeneous and the septum providing rather embryonic germinal zones [8, 14, 15, 16]. The newly spe- specifically the CR+ interneurons of the GL. Addition- cified neuroblasts migrate tangentially along the rostral ally, we show that Gsx2 is required for the expansion of migratory stream (RMS) to the OB, where they radially specified septal SVZ progenitors that give rise to CR+ migrate to populate different layers and undergo matur- interneurons. ation [17, 18]. Recently, it has been suggested that OB interneurons of distinct subtypes are produced by Methods progenitor cells in different topological domains of the Animals perinatal telencephalon [2, 7, 9, 10, 16]. For example, Olig2Cre/+ mice [35] and Gsx2e-CIE mice [36] were geno- TH+ interneurons were shown to be generated from the typed with the following primers: JaxCre-5′ (5′-GCGGTC LGE, whereas CR+ interneurons are suggested to be TGGCAGTAAAAACTATC-3′)andJaxCre-3′ (5′-CCAT predominantly produced by the septum [9, 10, 16]. GAGTGAACGAACCTGG-3′). Gsx2flox/+, Gsx2RA/+ and Despite the relatively detailed studies of the origins of Gsx2EGFP/+ alleles were genotyped as previously described OB interneuron subtypes, few genetic approaches have [31, 32]. RosatdTomato (Ai14) mice were genotyped with the been taken to address the contributions of different following primers: Rosa-tdTomato-5′ (5′-GGCATTAAAG- progenitor domains to OB interneuron diversity at CAGCGTATCC-3′)andRosa-tdTomato-3′ (5′-CTGTTC embryonic stages. CTGTACGGCATGG-3′) [37]. Zic3-lacZ BAC transgenic The normal generation and specification of OB inter- mice [38] were genotyped with the following primers: neurons are regulated by a number of transcription βgal5’ (5′-TGGGGAATGAATCAGGCCACGG-3′) and factors [7, 19]. For example, previous studies suggested βgal3’ (5′-GCGTGGGCGTATTCGCCAAGGA-3′). The that zinc finger transcription factor Sp8, which is Gsx1 knockout mice [39] and staged embryos were expressed by many post-mitotic neuroblasts from both genotyped with the following primers: Gsx1-WT1 Qin et al. Neural Development (2017) 12:13 Page 3 of 14

(5′-CGGGTGAAGCACAAGAAAGAAG-3′), Gsx1-WT2 field images were captured using an Olympus BX50 (5′-CCAATGGTCCTCTAAAAGGCG-3′), Gsx1-MT1 microscope. (5′-GGTTCATCATCACTAATCACGACG-3′)andGsx1- MT2 (5′-CGCTGTTCTCCCTCTTCCTCATCTC-3′). For embryonic analysis, the morning of the vaginal Generation of Gsx1 antibody plug observed was designated embryonic day (E)0.5. The Gsx1 antibody was raised in guinea pigs against the C- Embryos were fixed in 4% PFA overnight at 4 °C, exten- terminal peptide of human Gsx1, SAPQGCKCASLSSAKC- sively rinsed in PBS and cryoprotected in 30% sucrose in SEDDDELPMSPSSSGKDDRDLTVTP (service provided by PBS. Embryos were embedded in Neg-50 embedding Pierce Custom Services, a subdivision of Life Technologies/ medium for frozen tissue sectioning (Thermo Scientific) Thermo-Fisher Pierce) and used at 1:4000 dilution. Heated and coronal or horizontal sections were obtained at citrate retrieval solution was used to enhance the staining 12 μm on a cryostat. Sections were mounted onto of Gsx1. This antibody was generated in Dr. Ronald Wac- ’ ’ SuperFrost Plus Microscope Slides (Fisher Scientific) laws lab at Cincinnati Childrens Hospital Medical Center and stored at −20 °C until processed. Postnatal brains and its specificity was confirmed on Gsx1 mutants [39] at were collected at P14. Brains were removed from skull E18.5 (shown in Additional file 1: Figure S1). Control stain- and fixed in 4% PFA overnight at 4 °C before being ing for Gsx1 protein in the ventral most LGE progenitors extensively rinsed in PBS and cryoprotected in 20% and in the developing hypothalamus (Additional file 1: sucrose in PBS. Brains were then embedded in Neg-50 Figure S1) is similar to previously characterized Gsx1 gene (Thermo Scientific) and coronal sections were obtained expression [28, 30, 32, 41]. at 14 μm on a cryostat. Again sections were mounted onto SuperFrost Plus Microscope Slides (Fisher Bromodeoxyuridine (BrdU) labeling Scientific) and stored at −20 °C until staining. Pregnant females were given one dose of BrdU (Sigma- Aldrich) (100 mg/kg) by intraperitoneal injection with Immunohistochemistry embryos at E15.5 stage. Embryos were collected 1 h or Primary antibodies were used at the following concen- 24 h later to examine proliferation and cell cycle reten- trations: rabbit anti-βgal, 1:1000 (Biogenesis); goat anti- tion [42]. Embryos were processed as described above. βgal, 1:1000 (Biogenesis); chicken anti-βgal, 1:500 Cryosectioned 12 μm tissues were treated 50 min for (Abcam); rabbit anti-calbindin, 1:2500 (a gift from Dr. antigen-retrieval with 2 N HCl at room temperature Piers Emson, Babraham Institute); goat anti-calretinin, followed by PBS washes and incubation in rat anti-BrdU 1:2000 (Millipore); rabbit anti-Gsx2, 1:5000 [40]; rabbit (1:200, BioRad) overnight. anti-Ki67, 1:1000 (Abcam); rabbit anti-Mef2c, 1:2000 (Protein Tech Group); mouse anti-Neurofilament (NF- M), 1:200 (deposited to the Developmental Studies In situ hybridization Hybridoma Bank by T.M. Jessell and J. Dodd); rabbit In situ hybridization was performed as previously de- anti-Pax6, 1:1000 (Biolegend); goat anti-Sp8, 1:8000 scribed [43]. Digoxigenin-labeled antisense probe against (Santa Cruz Biotechnology); chicken anti-TH, 1:500 3′-UTR of Zic3 mRNA [38] was used on 12 μm cryosec- (Aves Labs), rabbit anti-panZic, 1:2000 (a gift from Dr. tions from E18.5 embryos. Stephen Brown, University of Vermont). Bright-field staining was obtained by using diaminobenzidine (DAB) as the chromogen following 2-h incubation in biotinyl- Quantification ated goat anti-guinea pig (1:200, Vector Laboratories), For the quantification of tdTomato fate mapped and horse anti-goat (1:200, Jackson Immunoresearch) or Zic3-lacZ expressing OB interneuron subtypes in the swine anti-rabbit (1:200, DAKO) and 1-h incubation in GL, three animals were analyzed on the medial side of ABC solution (Vector Laboratories). Secondary anti- the OB. Three control and three Gsx2 germline mutant bodies for fluorescent staining (Jackson Immunore- (KO) embryos were analyzed for Gsx2 KO phenotype. search) were donkey anti-rabbit antibodies conjugated Quantification of the embryonic (E18.5) OB phenotype with Alexa488, Cy3 or Alexa647, donkey anti-goat in Gsx2 conditional mutants (cKOs) was performed on antibodies conjugated with Alexa488, Cy3 or Alexa647, three controls and three Gsx2 cKOs. Quantification of donkey anti-chicken antibodies conjugated with Alexa488, the P14 OB phenotype in Gsx2 cKOs was performed Cy3 or Alexa647 and donkey anti-mouse antibody using Imaris (Bitplane) to analyze the GL on the medial conjugated with Cy3. Fluorescent slides were covered side of OB of controls (n = 5) and Gsx2 cKOs (n = 5). with Fluoromount-G (SouthernBiotech). DAB slides Quantitative results were presented as mean ± standard were covered with DPX (Sigma). Confocal images were error of the mean (s.e.m.). Statistical significance was taken on NikonA1RGaAsP inverted microscope. Bright determined using the Student’s t-test. Qin et al. Neural Development (2017) 12:13 Page 4 of 14

Results interneurons (Fig. 1J-O). Interestingly, few, if any, Zic3-lacZ marks a subpopulation of septal cells and their βgal+ cells were observed to express the GCL OB OB derivatives interneuron marker Mef2c [47, 48] (Fig. 1P). Thus, Zic (1-4) genes are highly expressed in medial telence- Zic3-lacZ mice provide a useful tool to study the OB phalic progenitors, including those in the septum, but interneuron progeny of septal progenitors. not in lateral progenitors, and have been implicated in OB interneuron development [44, 45]. Additionally, Differential contributions to OB interneuron subtypes recent tamoxifen-regulated fate mapping studies using from septal and LGE progenitors Fgf8CreER/+ and Fgf17CreER/+ alleles have revealed that, at OB interneurons are physiologically and biochemically early telencephalic stages, these medial telencephalic re- diverse and can be categorized into different subtypes gions give rise to the septum, as well as a subpopulation based on various criteria including the biochemical of OB interneurons [46]. To label septal progenitors and markers they express, OB layer they occupy as well as their progeny, we utilized a lacZ reporter under the neuronal connections they make [3, 4, 5, 6, 7, 19]. Previ- control of cis-regulatory modules of the Zic3 gene. The ous studies have suggested that the biochemical and Zic3-lacZ transgenic line was generated by inserting morphological diversity of OB interneurons is deter- lacZ into the Zic3 locus in a bacterial artificial chromo- mined by the location of the neural stem cell domains some (BAC) [38]. We observed that β-galactosidase from which they originate [9, 10, 16, 49]. To further (βgal) from Zic3-lacZ transgene was largely restricted to explore the OB interneuron subtypes derived from em- the Zic+ medial telencephalon, with only a few scattered bryonic and neonatal neural progenitor cells of distinct cells occasionally observed in the ventricular zone (VZ) domains, we utilized Zic3-lacZ transgene as a genetic of the rostroventral LGE (Fig. 1A-G). We also detected short-term fate map tool to study OB interneuron βgal staining in the striatum from E18.5 onward; subtypes from the septum. In addition, we recently gen- however, this staining marked axons likely from the di- erated the Gsx2e-CIE transgenic line, which robustly encephalon, as confirmed by Neurofilament-M double fate-maps LGE (but not septal) derivatives [36] and staining (Fig. 1C, inset and data not shown). In the med- therefore allows us to assess LGE-derived OB inter- ial progenitors, a gradient of βgal staining was noticeable neuron subtypes in the same tissue as the Zic3-lacZ la- in the septal VZ, which was more uniform in the dorsal beled interneurons. Indeed, we crossed the Gsx2e-CIE septum (Fig. 1A-G). Overall, the lacZ expression pattern mice with a RosatdTomato (Ai14) reporter line [37] in the was consistent with that of the endogenous Zic3 gene presence of Zic3-lacZ and found that LGE-lineage cells and overall Zic proteins (Fig. 1E-G) [44]. At E15.5, βgal fate-mapped by tdTomato (hereafter referred to as tdTo- was robustly expressed in the septum. Indeed, we found mato+ cells) and βgal from Zic3-lacZ were largely non- many Gsx2+ progenitor cells in the septal VZ coexpres- overlapping within the OB at P14, when the peak of sing βgal (Fig. 1A, B). Similarly, many Gsx2+ progenitor neonatal OB interneuron neurogenesis occurs [7, 15] cells were βgal+ in the E18.5 septum (Fig. 1C, D). These (Fig. 2A-E). Interestingly, although the βgal+ and tdTo- data suggested that Zic3-lacZ could be an effective tool mato+ cells were mixed within the GL of the OB, βgal+ to mark Gsx2+ progenitor cells in the septum and pos- and tdTomato+ neuroblasts in the RMS remained on the sibly their progeny. The septum is an important source side from which they originated (i.e. medial and lateral, of many neuronal cell types, including OB interneurons respectively) (Fig. 2A, inset). In the P14 OB, we found [9, 10, 14, 16, 33, 44, 46]. Moreover, Zic3 expressing cells βgal+ cells were largely confined to the GL, with only originating from the septum have been shown to migrate scattered βgal+ cells in other OB layers, including the ex- toward the OB [44]. To understand whether medial pro- ternal plexiform layer (EPL) and the GCL (Fig. 2B). In genitors in the septum contribute to the developing OB, fact, about 29.3 ± 1.3% of the GL cells were βgal+, we immunostained horizontal sections from E18.5 Zic3- whereas only 4.7 ± 1.0% of the cells in GCL expressed lacZ septum and OB with antibodies against βgal and βgal. This is in line with the observation that few, if any, Sp8 and found many βgal+ cells expressing Sp8 migrat- of the Mef2c+ granule cells were βgal+ in the E18.5 OB ing anteriorly from the septum to the OB in a pattern (Fig. 1P). In contrast, numerous LGE-derived tdTomato+ similar to Zic3 gene expression (inset in Fig. 1H). We cells were found in both the GL and GCL (Fig. 2C-E) also found many βgal+ cells in the germinal zone of the [36]. The GL enrichment of βgal+ cells suggested that E18.5 OB, with a strong bias towards its medial side; the GL was a major destination of septum-derived OB moreover, in the GL we found βgal+ cells distributed cells and therefore represented the focus of the present around the OB with a concentration on the medial study. To examine the neuronal subtype identity of the side (Fig. 1J-O). Many βgal+ cells in the E18.5 OB βgal+ cells in the GL, we immunostained the P14 OB were also labeled by panZic and expressed the tran- with antibodies against markers of periglomerular inter- scription factor Sp8, suggesting they were indeed OB neuron subtypes, CB, CR and TH and found virtually no Qin et al. Neural Development (2017) 12:13 Page 5 of 14

Fig. 1 βgal from Zic3-lacZ marks the septal primordia and OB cells originating from septum. (a-d) βgal was enriched in the E15.5 and E18.5 septum and co-localized with Gsx2 within VZ progenitors (b, d). Insets in (b)and(d) show high magnification views of septum (b’)and(d’). Inset in (c)showsβgal staining in the E18.5 striatum was found in axons ascending from diencephalon as evidenced by co-labeling with neurofilament (c’). (e-g) βgal from Zic3- lacZ displayed a dorsal to ventral gradient, similar to Zic proteins stained by panZic antibody, in the E18.5 septum. Boxes in (e) represent high magnification views in (f)and(g). (h, i) Horizontal section of E18.5 brain showed βgal+ cells migrating to the OB along the RMS and expressing Sp8 on the medial side (indicated by arrows) in a pattern similar to endogenous Zic3 gene expression shown in the inset (h’), whereas very few βgal+ cells were on the lateral side despite the presence of migrating Sp8+ cells (indicated by arrowheads). (J-L)Manyβgal+ and Zic+ cells were found in the E18.5 OB in regions including the forming GL (k) and the germinal zone (l). Boxes in (j) represent high magnification views in (k)and(l). Most βgal+ cells were Zic+, while many Zic+ cells were βgal−.(m-o)Manyβgal+ cells in the forming GL (n) and germinal zone (o) of the OB co-expressed Sp8, constituting a subpopulation of Sp8+ interneurons. Boxes in (m) represent high magnification views in (n)and(o). (p)Mef2c+ granule cells in the E18.5 OB did not express βgal

− CB+ neurons were labeled by βgal (Fig. 2F, L). In βgal+ cells were Sp8 and displayed glial morphology to- contrast, about 50.1 ± 3.0% of the CB+ cells were tdTo- gether with immunoreactivity for the astrocyte marker mato+ (Fig. 2G, L), suggesting that a significant portion GFAP (data not shown). The βgal+Sp8+ double positive of CB+ interneurons were derived from the LGE but not cells comprised about one third (36.0%) of the total Sp8+ the septum. Similarly, we found few, if any, TH+ (i.e. cells in the GL, whereas about 25.9 ± 2.0% of the Sp8+ GL dopaminergic) neurons were βgal+, whereas 40 ± 0.6% of neurons were tdTomato+ (i.e. LGE-derivatives) (Fig. 2J, L). them were fate-mapped by tdTomato (Fig. 2H, I, L), in- Many of the Sp8+ cells in the GL express the calcium dicating that TH+ OB interneurons are not septum- binding protein CR [19, 20], and CR+ interneurons have derived and that the LGE serves as an important source been suggested to arise predominantly from the for this neuronal subtype. The zinc finger transcription fac- septum at postnatal time points [9, 10]. Indeed, we tor, Sp8, is expressed by many interneurons residing in both found 22.2 ± 1.8% of the CR+ cells in the GL were the GL and GCL [20]. We found that 85.7 ± 2.6% of the βgal+, accounting for about 30.5 ± 4.0% of the βgal+ βgal+ cells in the GL were Sp8+ (Fig. 2J). A small portion of cells, supporting a septal origin for at least a portion Qin et al. Neural Development (2017) 12:13 Page 6 of 14

Fig. 2 Septum and LGE contribute to OB interneuron subtypes differently. (a) βgal+ (septum-derived) and tdTomato+ (LGE-derived) cells distributed around the P14 OB, despite their bias towards medial and lateral migration, respectively (inset of RMS (a’)). (b-e) βgal and tdTomato expressing cells were largely separated and showed distinct layer localization in the P14 OB. Boxes in (b)and(c) represent high magnification views in (d) and (e). βgal+ cells were enriched in the GL (b, d) whereas tdTomato+ cells were abundant in both GL and GCL (c-e). (f, g) LGE (tdTomato+) but not septum (βgal+) progenitors gave rise to CB+ PGCs. (h, i)TH+ PGCs did not originate from septum. Instead, many of them were generated from the LGE. (j, k) Both LGE (tdTomato+ cells indicated by arrows)andseptum(βgal+ cells indicated by arrowheads) contributed to Sp8+ and CR+ PGCs. Boxes in (j)and(k) represent high magnification views in (j’, j”, k’, k”) respectively. (l) Quantification of each PGC subtypes from LGE- versus septum-lineages. Data represent the mean ± s.e.m. of this OB interneuron subtype (Fig. 2K, L). However, been well characterized. To test the requirement of we also observed that about 17.6 ± 1.7% CR+ inter- Gsx2 in the generation of OB cells from septum, we neurons were tdTomato+,indicatingthatatleasta crossed the Zic3-lacZ allele onto Gsx2 germline subpopulation of these interneurons originate from knockout (Gsx2 KO, Gsx2RA/EGFP or Gsx2RA/RA)and the LGE (Fig. 2K, L). Taken together, these data control (Gsx2RA/+ or Gsx2EGFP/+)mice[31,32].We support the notion that the LGE, more specifically found reduced numbers of βgal+ cells in both the germinal thedLGE,givesrisetoallthreesubtypes(i.e.TH,CB zone and GL of the Gsx2 KO bulb at E18.5 (Fig. 3a, b). In and CR) of GL interneurons, while the septum largely agreement with previous studies [31, 32], we also observed contributes to the CR subtype. Moreover, the dLGE a 70% reduction (control: 96.9 ± 11.4 versus Gsx2 KO: provides neurons to populate both the GL and GCL, 26.8 ± 7.0 cells, p =0.006)ofSp8+ interneurons in the while the septum-derived progeny are specifically forming GL of the Gsx2 KO OB, which was concomitant targeted to the GL. with about a 60% loss (control: 21.3 ± 3.4 versus Gsx2 KO: 7.8 ± 3.3 cells, p =0.045)oftheβgal+ (i.e. septum-derived) Impaired septum-derived OB neurogenesis in germline population (Fig. 3c, d). These data supported the notion Gsx2 knockouts that Gsx2 is critical for the normal generation of septum- The homeobox gene Gsx2 is expressed at various derived OB interneurons as is the case for the LGE- levels by many VZ progenitor cells in the embryonic derived populations [31, 32]. ventral telencephalon, including the LGE, MGE and septum [29, 40, 50], as well as in the postnatal Reduced septum-derived interneurons in the Olig2Cre/+ dorsolateral SVZ (dlSVZ), a derivative of the embry- driven Gsx2 cKO OB onic dLGE [48]. Previous studies revealed that Gsx2 As shown above, reduction in the numbers of βgal+ and is critical for the normal generation of many LGE- Sp8+ cells was observed in the E18.5 Gsx2 mutant OB. derived cell types including OB interneurons, amyg- However, germline Gsx2 mutant mice die at birth [52], dalar intercalated cells and striatal projection neurons preventing further analysis of the impact on the gener- [20, 27, 28, 29, 30, 31, 32, 40, 51]. Despite that ation of mature OB phenotypes. Olig2 is robustly altered transcriptional profiles have been reported in expressed not only by cells of the oligodendrocyte the Gsx2-deficient septum [34], the role of Gsx2 in lineage [53], but also by many neurogenic progenitor specifying septum-derived OB interneurons has not cells in the VZ of ventral telencephalic regions including Qin et al. Neural Development (2017) 12:13 Page 7 of 14

Fig. 3 Gsx2 germline knockout (KO) impairs the generation of septum-derived OB cells. a, b Gsx2 KO carrying the Zic3-lacZ allele showed a dramatic loss of βgal+ cells in the E18.5 OB. c, d Septum-derived (i.e. βgal+) and non-septum-derived (i.e. βgal−)Sp8+ interneurons were also severely compromised in the E18.5 Gsx2 KO OB the MGE, LGE and the septum [54, 55, 56]. Within the antibodies against Sp8 and βgal. In agreement with the LGE, Olig2 is highly expressed in the VZ cells of the observation in E18.5 Gsx2 KO OB, we found the number ventral (v)LGE while its expression in the dLGE is quite of βgal+ cells was reduced by 73% in the Gsx2 cKO OB, as limited. Therefore we took advantage of an Olig2Cre/+ compared to controls (control: 26.1 ± 2.6 versus cKO: line [35] to selectively knockout Gsx2 in the septum and 7.1 ± 2.0 cells per field, p = 0.001) (compare Fig. 5B with vLGE, while leaving it largely intact within the dLGE, as A). The total number of Sp8+ cells in the Gsx2 cKO GL previously published [57]. We found a complete loss of was reduced by 31% (control: 70.4 ± 3.8 versus cKO: Gsx2 in the E15.5 Olig2Cre/+;Gsx2fx/fx (Gsx2 cKO) septum 48.3 ± 3.3 cells per field, p = 0.023) (Fig. 5A-C). Import- and vLGE (Fig. 4B) in comparison to Olig2Cre/+;Gsx2fx/+ antly, the number of Sp8+βgal+ double labeled cells (i.e. (control) which show robust Gsx2 expression in septum septum-derived) in the Gsx2 cKO GL was reduced by ap- and throughout the LGE (Fig. 4A). The expression of proximately 80% from control (control: 16.7 ± 1.1 versus Gsx2 in the dLGE of the Gsx2 cKOs was largely intact cKO: 3.3 ± 1.4 cells per field, p = 0.003) (Fig. 5A-C). In − (Fig. 4B). We observed a robust upregulation of the closely contrast, the number of Sp8+βgal GL cells in the Gsx2 related family member, Gsx1, in the Gsx2 cKO septum cKO OB, which are presumably derived largely from the and vLGE (Fig. 4C, D). This is in line with previous studies dLGE, was not significantly different from that in the that show Gsx1 can partially compensate for the loss of control (control: 53.7 ± 4.3 versus cKO: 45.0 ± 3.5 cells Gsx2 in the LGE [28, 30]. To investigate whether medial per field, p = 0.11) (Fig. 5A-C). derived OB interneurons are compromised when Gsx2 is At E18.5 the OB is undergoing development, and inactivated in the septum, we used Zic3-lacZ as a reporter many interneurons that are specified during embryonic and immunostained E18.5 control and Gsx2 cKO OB with stages have not yet reached their final destination in the Qin et al. Neural Development (2017) 12:13 Page 8 of 14

Fig. 4 Conditional inactivation of Gsx2 in the septum by Olig2Cre/+.(a, b) Gsx2 protein was lost in the septum and vLGE of the E15.5 Gsx2 cKO embryos, however its expression was relatively normal in the Gsx2 cKO dLGE. Insets in (a) and (b) represent low magnification DAPI stains (a’, b’). Dashed boxes in (a’) and (b’) represent the magnification in (a) and (b). (c, d) Accordingly, the Gsx2 family member, Gsx1 was found upregulated in the E15.5 Gsx2 cKO septum and vLGE bulb and begun to express mature biochemical markers Gsx2 cKO OB (Fig. 5D-F). Furthermore, the compromised (e.g. CB, CR and TH). In addition, only a portion (about Sp8+ population was primarily due to the loss of septum- 20%) of the OB interneurons in the rodent are born em- derived Sp8+ periglomerular cells, as a 36% reduction (con- bryonically [15, 58], a significant amount of them are trol: 506.1 ± 30.5 versus cKO: 324.5 ± 18.4 cells/mm2, generated during the early postnatal stage [7, 15]. Unlike p = 0.001) of Sp8+βgal+ double labeled cells in the GL was − Gsx2 germline knockouts, Olig2Cre/+;Gsx2fx/fx (Gsx2 observed (Fig. 5D-F). In contrast, the Sp8+βgal GL cells cKO) mice are viable after birth, allowing us to assess (presumably dLGE-derived) in the mutants were not interneuron phenotypes in the postnatal OB. Therefore significantly different from control (control: 1481.7 ± 40.0 we generated P14 Olig2Cre/+;Gsx2fx/+;Zic3-lacZ (control) versus cKO: 1367.3 ± 50.2 cells/mm2, p = 0.11) (Fig. 5D-F). and Olig2Cre/+;Gsx2fx/fx;Zic3-lacZ (Gsx2 cKO) mice and im- ThepostnatalseptumisknowntogiverisetoCR+ inter- munostained their OB with antibodies against different OB neurons in the GL (Fig. 2K, L) [9, 10, 16]. We also detected interneuron markers. We found a 15% reduction (control: a 13% reduction (control: 849.9 ± 26.7 versus cKO: 1987.8 ± 54.4 versus cKO: 1691.8 ± 67.5 cells/mm2, 741.5 ± 34.2 cells/mm2, p = 0.037) of CR+ interneurons in p = 0.009) of Sp8+ interneurons along with a significant re- the GL of the Gsx2 cKO OB (Fig. 5G-I). Importantly, a duction (35%) of βgal+ cells (control: 595.3 ± 32.1 versus 29% reduction of the septum-derived CR+βgal+ cells was cKO: 387.7 ± 17.3 cells/mm2, p = 0.0005) in the GL of the observed in the GL of the Gsx2 cKOs (control: Qin et al. Neural Development (2017) 12:13 Page 9 of 14

Fig. 5 Impairment of septum-derived PGCs in the Gsx2 cKO OB. (a-c) Septum-derived GL cells marked by βgal were reduced, leading to a significant loss of septum-originated Sp8+ interneurons (i.e. βgal+) and a milder reduction of total Sp8+ PGCs in the E18.5 Gsx2 cKO OB, whereas LGE-derived Sp8+ interneurons (i.e. βgal−) were largely normal. Boxes in (a)and(b) indicate the GL area shown in (a’)and(b’) respectively. (d-f)Sp8+ PGCs were reduced in the P14 Gsx2 cKO OB, primarily due to the reduced number of the septum-derived (i.e. βgal+) interneurons. Arrowheads indicate Sp8+βgal+ cells that originated from the septum. (g-i)P14Gsx2 cKO OB showed reduced number of CR+ PGCs, particularly those βgal-expressing ones generated from the septum. Other CR+ PGCs (i.e. βgal−), presumably originating from other regions including LGE, were largely intact. Data represent the mean ± s.e.m. *p <0.05

171.2 ± 13.5 versus cKO: 121.7 ± 9.2 cells/mm2, p = 0.016), control and Gsx2 cKO embryos. Similar to what was − whereas the CR+βgal population (i.e. dLGE-derived) was found in the Gsx2-deficient LGE [28], we observed a not significantly different from that in control (control: dramatic loss of Sp8+ cells in the Gsx2 cKO septum, 678.7 ± 31.9 versus cKO: 619.8 ± 27.7 cells/mm2, p =0.2) particularly at the rostral level where a large amount of (Fig. 5G-I). Few, if any, of the CB+ or TH+ interneurons in Sp8+ cells were present in the control septum (arrow- the GL originate from the septum (see Fig. 2F, H, L). Ac- heads in Fig. 6). In contrast, Sp8 staining in the Gsx2 cordingly, normal numbers of CB+ and TH+ GL interneu- cKO dLGE was comparable to that in the control em- rons were observed in the Gsx2 cKO OB (data not shown). bryos (arrows in Fig. 6), consistent with the fact that Gsx2 expression remains in the dLGE of conditional Conditional knockout of Gsx2 in the septum leads to mutants. Gsx2 is critical for maintaining LGE progenitor development defects cells in an undifferentiated state and promoting their Gsx2 has been implicated in the generation of OB inter- self-renewal capacity [50]. In the absence of Gsx2, the neurons from the dLGE by regulating the temporal embryonic LGE displays compromised cell proliferation specification of Sp8+ neuroblasts [20, 31, 32, 50, 57]. To and, thus, fails to establish a normal SVZ [28]. To inves- determine whether Gsx2 inactivation by Olig2Cre/+ im- tigate whether Gsx2 regulates proper cell proliferation in pairs the normal specification and/or generation of Sp8+ the developing septum, we pulsed pregnant females with neuroblasts in the developing septum, we analyzed E15.5 one dose of BrdU at E15.5 and collected embryos 1 h Qin et al. Neural Development (2017) 12:13 Page 10 of 14

suggesting the impairment of the formation of the sec- ondary proliferative zone (i.e. SVZ) in the Gsx2-deficient septum. To determine whether cell cycle exit is altered in the Gsx2 cKO septum, one dose of BrdU was given at E15.5 and embryos were collected 24 h later [42]. Again, we performed double immunofluorescent staining for BrdU and Ki67 and calculated cell cycle retention index, measured by the percentage of BrdU+ cells labeled at E15.5 remaining in cell cycle (i.e. BrdU+Ki67+/BrdU+), in both control and Gsx2 cKO septum. While the cell cycle retention index in the VZ of control and Gsx2 cKO septum were comparable (control: 29.4% versus cKO: 31.6%, p = 0.26) at E16.5, we found a reduced percentage of BrdU+ cells remaining in the cell cycle (i.e. Ki67+) within the SVZ of the Gsx2 cKO septum (control: 38.4% versus cKO: 25.1%, p = 0.0006) (Fig. 7e-g). These findings indicate that Gsx2 is required for the correct establishment of a proliferative SVZ in the septum. Moreover, proliferative progenitors in the Gsx2-deficient septal SVZ appear to exit the cell cycle prematurely, thus limiting the number of septum-derived OB inter- neurons, which is in line with the observed OB pheno- type in Gsx2 germline and cKO mutants.

Discussion In this study, we investigated the regional contributions to OB interneuron diversity at embryonic and neonatal stages using both long-term and short-term genetic fate- mapping approaches. Our results indicate that the septum contributes to subpopulations of Sp8+ and CR+ interneurons in the GL but not to their CB+ or TH+ counterparts. In contrast, the dLGE contributes to all these subtypes, including CB+,CR+ and TH+, as well as large numbers of interneurons that occupy both the GL and GCL. The homeobox gene Gsx2 has been suggested to be critical for the normal generation of OB interneu- rons from the embryonic dLGE [31, 32], however, its role in the generation of septum-derived OB interneu- rons has not been described. By conditionally inactivat- Fig. 6 Reduced neuroblasts in the Gsx2 cKO septum. The number of ing Gsx2 in the septum while largely preserving its Sp8+ neuroblasts in the E15.5 Gsx2 cKO septum was impaired, mostly expression in the dLGE, we found reduced numbers of at a rostral levels (compare (a, c)to(b, d)indicatedbyarrowheads), septum-derived Sp8+ and CR+ OB interneurons in the whereas Sp8 staining in the caudal septum was relatively normal + GL. In addition, our data suggest that the OB inter- (compare (e)and(f), indicated by arrowheads). In contrast, Sp8 + neuroblasts generated in the LGE were largely unchanged (indicated neuron defects (reduction of CR GL cells) observed in by arrows from (a-f)) Gsx2 cKO animals result from the impaired proliferation of OB interneuron progenitors in the septal SVZ. OB interneurons are generated from both dLGE and after BrdU administration to label cells in S-phase. septum embryonically [8, 14, 16, 20, 31, 33]. It has been Double staining for BrdU and the cell proliferation suggested that OB interneurons of different subtypes marker Ki67 demonstrated that cell proliferation in the originate from distinct progenitor/neural stem cell septal VZ of Gsx2 cKO embryos was indistinguishable domains in the postnatal telencephalon [9, 10, 16]. from that in the control (Fig. 7a-d). However, the num- Although many studies have attempted to address this bers of Ki67+ and BrdU+ cells in the SVZ were dramatic- notion, few genetic approaches have been taken to ally reduced in the Gsx2 cKO septum (Fig. 7a-d), characterize the origins of OB interneuron subtypes Qin et al. Neural Development (2017) 12:13 Page 11 of 14

Fig. 7 Impaired cell proliferation in the Gsx2 cKO septum. a-d Gsx2 cKO septum showed reduced cell proliferation in the SVZ, but not in the VZ, at E15.5, as revealed by Ki67 and BrdU staining after one-hour BrdU labeling. The VZ was defined according to the apical ventricular surface and the basal region where S-phase BrdU+ nuclei were enriched. The same size box was placed adjacent to the VZ to represent the SVZ. e-g Cell cycle retention index was relatively normal in the VZ but decreased in the SVZ of the Gsx2 cKO septum 24 h after E15.5 BrdU administration. Data represent the mean ± s.e.m. *p <0.05,**p < 0.01, ***p <0.005 generated during embryonic and the subsequent were βgal+ (data not shown). In the OB, βgal signal neonatal periods, when many OB interneurons are born represents both persistent and down-regulated Zic gene [7, 15]. Recently, a pan-antibody against Zic proteins expression. Most of the βgal+ cells were panZic+, but was used to identify OB interneurons derived from some of the cells in which βgal was persistent were not septum [10]. Although panZic+ cells were restricted in and thus likely represent a short-term fate map of the the E18.5 septum and most of the βgal+ cells from the Zic lineage. Thus, Zic3-lacZ appears to be a reliable Zic3-lacZ lineage were also panZic+ in the OB GL, we short-term fate map tool for studying septal cells and detected some panZic+ cells in the P14 dlSVZ (data not their OB derivatives. shown). While panZic staining is enriched in the septum By combining the Zic3-lacZ allele together with the and remains a useful tool to examine septum derived recently characterized LGE driver Gsx2e-CIE [36], we cells in postnatal OB, we took a more specific approach assessed the unique contributions to the OB interneuron using Zic3 reporter mice expressing βgal from a Zic3 populations from septum and LGE separately. Our BAC construct [38]. We found βgal expression to be results, at both E18.5 and P14, indicate that septum largely restricted to the medial side of the telencephalon, primarily contributes to OB interneurons (i.e. βgal+)in including the septum, with only occasional clones ob- the GL, whereas dLGE-derived interneurons (i.e. tdTo- served in the ventral-most portion of the embryonic mato+) were observed to populate all OB layers, includ- LGE. In addition, no ectopic βgal signal was found in ing the GL and GCL. Furthermore, our findings indicate the P14 dlSVZ (data not shown). It was noticeable that that septum-derived OB progenitors are rather limited βgal from the Zic3-lacZ transgene showed a dorsal-high in potential to generate Sp8+ and CR+ cells in the GL, to ventral-low gradient in the septum, and although this whereas the dLGE progenitors give rise to all three pattern was consistent with that of endogenous Zic major GL subtypes, namely CB+,TH+ and interestingly proteins, its expression was mosaic. Despite this, a also a subpopulation of CR+ periglomerular cells. Merkle considerable portion of the Sp8+ cells in the septal SVZ et al. [9, 10] have previously suggested that the septum Qin et al. Neural Development (2017) 12:13 Page 12 of 14

represents an exclusive source for CR+ OB interneurons. periglomerular cells. Interestingly, the reduction of These studies examined the postnatal contributions of septum-derived Sp8+ interneurons in the P14 Gsx2 cKO septal progenitors, while our study includes both embry- OB was not as pronounced as that in the E18.5 OB. This onic and early postnatal time points. We found that the is probably due to the upregulation of Gsx1, a family septum and dLGE lineages both contribute to Sp8+ OB member of Gsx2, which has been shown to partially interneurons, among which about half also express CR compensate for the loss of Gsx2 in the LGE via upregu- [20]. In addition, CR+ cells have been detected in the lation at the VZ/SVZ boundary [28, 30, 32, 50, 57]. In- E18.5 dLGE, and similar stage Ascl1 mutants which deed, Gsx1 upregulation was already detected in E15.5 show aberrant Gsx2 and Sp8 expression in the dLGE, embryos also at the VZ/SVZ boundary of the Gsx2 cKO also exhibit enhanced CR staining in the dLGE and OB septum. In addition, Gsx-independent neurogenesis may [32]. The notion that the dLGE represents a separate also occur in the septum during later developmental source of CR+ OB interneurons is also supported by our stages. In fact, Gsx1 does not compensate for the loss of − findings that many CR+ (βgal ) interneurons were Gsx2 in the adult dlSVZ [48], suggesting a Gsx-inde- present in the Gsx2 cKO mice, in which Gsx2 remained pendent neurogenesis program for some OB interneuron largely intact in the dLGE. In fact, Zic3-lacZ (i.e. septal) subtypes. We observed reduced Sp8+ cells and impaired and Gsx2e-CIE (i.e. dLGE) lineages together account for cell cycle kinetics in the SVZ of the Gsx2-deficient only about 40% of the total CR+ interneurons in the GL. septum of E15.5 embryos. Similar phenotypes were also While this could be due, in part, to incomplete recom- reported in the Gsx2 knockout LGE [20, 28, 31, 32, 57]. bination/transgene expression, it is also possible that Interestingly, we found a reduced cell cycle retention other telencephalic regions may provide a separate index in SVZ but not in VZ progenitors of the Gsx2 subpopulation of CR+ OB interneurons. For example, a cKO septum. Given that Gsx2 is present in primary previous study using a transplantation approach progenitor cells (i.e. VZ), our results suggest that Gsx2 suggested that the pallium is able to contribute to CR+ regulates OB interneuron generation from the septum OB interneurons [2]. by enhancing indirect neurogenesis and/or indirectly The specification of diverse OB interneuron subtypes influencing the proliferation capacity of basal (i.e. SVZ) has been suggested to be the result of combinatory effects progenitors derived from Gsx2 expressing VZ cells. One of many transcription factors referred as transcription possible mechanism is that Gsx2 may repress or reduce factor codes [19, 59]. Our findings that both dLGE and Gsx1 expression. Despite that Gsx1 shares similar func- septum give rise to CR+ interneurons in the GL which are tion with Gsx2 in telencephalic patterning, it promotes distinguished, in part, by the expression of Zic proteins in progenitor maturation and neurogenesis while Gsx2 the septum-derived subpopulation raises the question helps to maintain progenitors in an undifferentiated whether CR+ interneurons with distinct telencephalic state [50]. Therefore, the Gsx2-deficient primary progen- origins are phenotypically/physiologically different. itors with upregulated Gsx1 may bias towards direct In the embryonic ventral telencephalon, Gsx2 is highly neurogenic cell division and give rise to SVZ cells that expressed by progenitor cells in the VZ of the septum exhibit reduced proliferative capacity. and LGE with a high dorsal to low ventral gradient in each region. We noticed co-localization of βgal from the Conclusions Zic3-lacZ transgene and Gsx2 in the septal VZ as well This study provides evidence that the embryonic/neonatal as maintained βgal expression in the embryonic and septum and dLGE contribute to OB interneuron diversity postnatal OB which allowed us to use this reporter differently. Specifically, the septum contributes CR+ cells mouse as a short-term genetic fate map tool to study the to the GL, while the dLGE gives rise to interneurons that contribution of Gsx2+ progenitors in the septum to their occupy both the GCL and GL as well as the 3 main sub- OB progeny. We found that septum-derived (i.e. βgal- types of GL interneurons (CB+,CR+ and TH+). Moreover, expressing) Sp8+ and CR+ OB interneurons were our findings indicate a role for Gsx2 in septal VZ progeni- reduced in the GL when Gsx2 was selectively inactivated tors for the generation of proliferative SVZ progenitors in the septum using an Olig2Cre/+ driver. Similarly, we specified to generate CR+ GL interneurons in the OB. observed decreased panZic-labeled Sp8+ cells in the forming GL of the E18.5 Gsx2 cKO OB (data not shown). Moreover, this phenotype was not due to a lack Additional file of Zic proteins or βgal expression, as both remained in Additional file 1: Figure S1. Characterization of Gsx1 specific antibody. the Gsx2 cKO septal progenitors (data not shown). Immunostaining for Gsx1 in the telencephalon and diencephalon reveals Therefore, these findings indicate an important role for positive cells in the ventral most LGE and developing hypothalamus +/+ Gsx2 in regulating normal OB interneuron generation (Gsx1 ) at E18.5 (A, C). No Gsx1 positive cells are detected in Gsx1 mutant (Gsx1−/−) forebrain regions (B, D). (TIFF 9369 kb) from the septum, specifically the septum-derived CR+ Qin et al. Neural Development (2017) 12:13 Page 13 of 14

Abbreviations 5. Schoppa NE, Urban NN. Dendritic processing within olfactory bulb circuits. BAC: Bacterial artificial chromosome; BrdU: Bromodeoxyuridine; CB: Calbindin; Trends Neurosci. 2003;26:501–6. cKO: Conditional knockout; CR: Calretinin; dLGE: Dorsal lateral ganglionic 6. Wachowiak M, Shipley MT. Coding and synaptic processing of sensory eminence; E: Embryonic day; GCL: Granule cell layer; GL: Glomerular layer; information in the glomerular layer of the olfactory bulb. Semin Cell Dev LGE: Lateral ganglionic eminence; MGE: Medial ganglionic eminence; Biol. 2006;17:411–23. OB: Olfactory bulb; PGC: Periglomerular cell; RMS: Rostral migratory stream; 7. Lledo PM, Merkle FT, Alvarez-Buylla A. Origin and function of olfactory bulb SVZ: Subventricular zone; TH: Tyrosine hydroxylase; VZ: Ventricular zone; interneuron diversity. Trends Neurosci. 2008;31:392–400. βgal: β-galactosidase 8. Stenman J, Toresson H, Campbell K. Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and Acknowledgements olfactory bulb neurogenesis. J Neurosci. 2003;23:167–74. We thank Drs. Tom Jessell and Ben Novitch for the Olig2Cre/+ mice. We also 9. Merkle FT, Mirzadeh Z, Alvarez-Buylla A. Mosaic organization of neural stem thank Drs. Piers Emson and Stephen Brown for providing antibodies. cells in the adult brain. Science. 2007;317:381–4. 10. Merkle FT, Fuentealba LC, Sanders TA, Magno L, Kessaris N, Alvarez-Buylla A. Funding Adult neural stem cells in distinct microdomains generate previously This work is supported by the National Institutes of Health (NIH) grant unknown interneuron types. Nat Neurosci. 2014;17:207–14. R01NS044080 to K.C. 11. Blanchart A, De Carlos JA, López-Mascaraque L. Time frame of mitral cell development in the mice olfactory bulb. J Comp Neurol. 2006;496:529–43. Availability of data and materials 12. Imamura F, Ayoub AE, Rakic P, Greer CA. Timing of neurogenesis is a Data sharing not applicable to this article as no datasets were generated or determinant of olfactory circuitry. Nat Neurosci. 2011;14:331–7. analyzed during the current study. 13. Imamura F, Greer CA. Pax6 regulates Tbr1 and Tbr2 expressions in olfactory bulb mitral cells. Mol Cell Neurosci. 2013;54:58–70. Authors’ contributions 14. Long JE, Garel S, Alvarez-Dolado M, Yoshikawa K, Osumi N, Alvarez-Buylla SQ and RRW performed the experiments. SMW provided essential reagents. RJL. Dlx-dependent and -independent regulation of olfactory bulb SQ, RRW and KC conceived of the study, and participated in its design and interneuron differentiation. J Neurosci. 2007;27:3230–43. data interpretation. SQ drafted the manuscript and SMW, RRW and KC edited 15. Batista-Brito R, Close J, Machold R, Fishell G. The distinct temporal origins of and revised the manuscript. 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J Mol Histol. 2007;38:517 25. The authors declare that they have no competing interests. 20. Waclaw RR, Allen ZJ 2nd, Bell SM, Erdélyi F, Szabó G, Potter SS, Campbell K. The zinc finger transcription factor Sp8 regulates the generation and diversity of olfactory bulb interneurons. Neuron. 2006;49:503–16. Publisher’sNote 21. Li X, Sun C, Lin C, Ma T, Madhavan MC, Campbell K, Yang Z. The Springer Nature remains neutral with regard to jurisdictional claims in transcription factor Sp8 is required for the production of parvalbumin- published maps and institutional affiliations. expressing interneurons in the olfactory bulb. J Neurosci. 2011;31:8450–5. 22. Ragancokova D, Rocca E, Oonk AM, Schulz H, Rohde E, Bednarsch J, Author details Feenstra I, Pennings RJ, Wende H, Garratt AN. TSHZ1-dependent gene 1Divisions of Developmental Biology, Cincinnati Children’s Hospital Medical regulation is essential for olfactory bulb development and olfaction. 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Proc Natl Acad Sci U S A. 2011;108:1675–80. and we will help you at every step: 51. Waclaw RR, Ehrman LA, Pierani A, Campbell K. Developmental origin of the neuronal subtypes that comprise the amygdalar fear circuit in the mouse. J • We accept pre-submission inquiries – Neurosci. 2010;30:6944 53. • Our selector tool helps you to find the most relevant journal 52. Szucsik JC, Witte DP, Li H, Pixley SK, Small KM, Potter SS. Altered forebrain • We provide round the customer support and hindbrain development in mice mutant for the Gsh-2 homeobox gene. Dev Biol. 1997;191:230–42. • Convenient online submission 53. Lu QR, Yuk D, Alberta JA, Zhu Z, Pawlitzky I, Chan J, McMahon AP, Stiles CD, • Thorough peer review Rowitch DH. Sonic hedgehog–regulated oligodendrocyte lineage genes • Inclusion in PubMed and all major indexing services encoding bHLH proteins in the mammalian central nervous system. Neuron. 2000;25:317–29. • Maximum visibility for your research 54. 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